1. Field of the Invention
The present invention is related to genes encoding novel neutral cellulases and compositions containing the novel neutral cellulases. These compositions are especially useful in the textile, detergent and pulp and paper industries.
2. Related Art
Cellulose is a linear polysaccharide of glucose residues connected by xcex2-1,4 linkages. In nature, cellulose is usually associated with lignin together with hemicelluloses such as xylans and glucomannans. The practical use of cellulases has been hampered by the nature of the known cellulases, which are often mixtures of cellulases having a variety of activities and substrate specificities. For that reason, it is desirable to identify sources from which cellulases having only the desired activities may be obtained.
A wide variety of cellulases are known in the art, most of which are acid cellulases. However, some neutral and alkaline cellulases have also been identified. Celluzyme(copyright) is a commercially-available cellulase preparation from Humicola insolens (Novo Nordisk, A/S). GB 2,075,028 and EP 406,314 describe the use of a Humicola insolens cellulase as an enzymatic additive in a wash detergent to reduce the harshness (stiffness) of cotton-containing fabrics. The cloning of a cellulase containing endoglucanase activity from Humicola insolens is described in WO 93/11249 and EP 531,372. EP 510,091 describes a cellulase from Bacillus spp. NCIMB 40250 that is useful in detergent compositions. EP 220,016 describes cellulases that are useful as clarification agents for colored fabrics. WO 94/07998 describes modified cellulases that possess an improved alkaline activity. WO 95/02675 describes detergent compositions that contain two different cellulases: a first cellulase that is catalytically amenable to particulate soil removal, and a second cellulase that is catalytically amenable to color clarification. WO 92/18599 describes a detergent preparation that contains both a cellulase and a protease. Cellulases have also been used industrially as an aid for the removal of printing paste thickener and excess dye after textile printing (EP 576,526).
EP 383 828 describes granular detergent compositions, which contain surface-active agent, a fabric-softening clay material, and cellulase granulates containing calcium carbonate. U.S. Pat. No. 5,433,750 describes detergent compositions containing a surface active agent, a builder system, a softening clay, a clay flocculating agent and a high activity cellulase, preferably Humicola insolens cellulase. U.S. Pat. No. 5,520,838 describes granular detergent compositions, comprising surface-active agent, a builder and a cellulase, preferably a Humicola insolens cellulase, said compositions being in a compact form, having a relatively high density and containing a low amount of inorganic filler salt.
Cellulase enzymes are used in a wide variety of industries in addition to the textile industry. For example, cellulases are used industrially for the deinking of newspapers and magazines (EP 521,999), for improving the drainage of pulp (WO 91/14822, WO 91/17243), and as a treatment for animal feed.
The unique properties of each cellulase make some more suitable for certain purposes than others. While the enzymes differ in a number of ways, one of the most important difference is pH optimum. Neutral cellulases have useful cellulase activity in the pH range 6-8, alkaline cellulases have useful cellulase activity in the pH range 7.5-10. Acid cellulases are active in the range of pH 4.5-6, but have little cellulase activity at higher pH values.
Neutral and acid cellulases are especially useful in the textile industry Klahorst, S. et al., Textile Chemist and Colorist 26:13-18, 1994; Nilsson, T. E., Aachen Textile Conference, DWI Reports 114:85-88 (1995); Videbxc3xa6k, T. et al, ITB Dyeing/Printing/Finishing, January 1994, pp. 25-29; Klahorst, S. et al., AATCC Int. Conf and Exhibit, Oct. 4-7, 1992, p. 243, Atlanta, Ga.; Kochavi, D. et al., Am. Dyestuff Resporter, September 1990, pp. 26-28; Tyndall, R. Michael, Textile Chemist and Colorist 24:23 (1992); Lange, N. K., in Proc. Second TRICEL Symp. on Trichoderma reesei Cellulases and Other Hydrolases, Espoo, Finland, 1993, ed. P. Suominen et al., Foundation for Biotechnical and Industrial Fermentation Research vol. 8, 1993, pp. 263-272. When used to treat fabric, cellulases attack the chains of cellulose molecules that form the cotton fibers, thereby affecting the characteristics of the fabric.
Traditionally, in xe2x80x9cstonewashing,xe2x80x9d pumice stones have been used to change the characteristics of the fabric. Gradually, cellulases are replacing pumice stones, which also give the fabric its desired final look but can cause damage to the machines, garments and sewage processing equipment. U.S. Pat. Nos. 4,832,864, 4,912,056, 5,006,126, 5,122,159, 5,213,581 and EP 307,564 disclose the use of cellulases in biostoning.
Cellulases are especially useful for stonewashing denim dyed with indigo as the dye mostly stays on the surface of the yarn and does not penetrate the fibers well. When used to treat cotton fabric, neutral cellulases generally require a longer wash time than do the acid cellulases. However, available neutral cellulases are less aggressive (active) against cotton than acid cellulases, and are reported not to compromise the strength of the fabric as readily as acid cellulases. Neutral cellulases have a broader pH profile and thus the pH increase that occurs during biostoning has little effect on the activity of the neutral enzyme.
The use of acid cellulases is hampered by their tendency to promote backstaining and a weakening of fabrics. In addition, the pH must be adjusted to to a range suitable for the function of the acid cellulases. Consequently, there is a clear demand for neutral cellulase enzyme preparations that do not cause backstaining or weakening of fabrics.
While it has become popular to use cellulases in the textile industry, simply changing the cellulase mixture that is used may produce a different finish. These problems have focused increasing attention on the search for reproducible mixtures of cellulases with desired properties. Thus there is a clear demand especially in the textile and detergent industry for novel cellulases active at neutral and alkaline pH values, not compromising the strength of fabrics, with good cleaning and/or fabric care and harshness reducing properties.
Recognizing the importance of identifying enzymes useful in textile biofinishing and biostoning and in detergent applications, the inventors have screened fungal species for neutral and alkaline cellulases with enzymatic characteristics that would be useful in such technologies.
These studies have resulted in novel cellulases originating from the genera Myceliophthora, Myriococcum, Melanocarpus, Sporotrichum and Chaetomium.
The invention is further directed to the spent culture medium or enzyme preparations prepared from the native hosts producing such novel cellulases.
The invention is further directed to the use of such culture medium or the use of such enzyme preparations in the textile and detergent industry and in the pulp and paper industries.
These studies have further resulted in the identification of three novel cellulases that are especially useful in the textile and detergent industry. Purified preparations from Melanocarpus sp. or Myriococcum sp. have revealed a 20 kDa cellulase with endoglucanase activity (designated herein as xe2x80x9c20K-cellulasexe2x80x9d), a 50 kDa cellulase (xe2x80x9c50K-cellulasexe2x80x9d), and a second 50 kDa cellulase (xe2x80x9c50K-cellulase Bxe2x80x9d). A novel gene product with high homology to the cellulase family, herein called xe2x80x9cprotein-with-CBDxe2x80x9d (where CBD means xe2x80x9ccellulose binding domainxe2x80x9d) was also discovered.
It is an object of the invention to provide enzyme preparations that contain one or more of the novel cellulases of the invention, especially the 20K-cellulase, the 50K-cellulase, the 50K-cellulase B and/or the protein-with-CBD.
It is a further object of this invention to provide a method for using such preparations for the finishing of textiles, especially the biostoning of denim, for the use said preparations in detergent compositions, and especially methods that use the 20K-cellulase, the 50K-cellulase, the 50K-cellulase B and/or the protein-with-CBD.
The invention is also directed to other neutral and/or alkaline cellulases having one or more of the amino acid sequences as described herein.
The invention is further directed to the genes encoding the 20K-cellulase, 50K-cellulase, 50K-cellulase B and the protein-with-CBD.
The invention is further directed to novel expression vectors comprising such genes and to novel hosts transformed with the vectors, especially hosts that are capable of high levels of expression of the proteins encoded by such genes.
The invention is further directed to the spent culture medium of such transformed hosts, the culture medium containing the novel 20K-cellulase, 50K-cellulase, the 50K-cellulase B and/or the protein-with-CBD, or enzyme compositions (enzyme preparations) containing one or more of these proteins that have been prepared from such culture media.
The invention is further directed to the use of such culture medium or the use of such enzyme preparations in the textile and detergent industry and in the pulp and paper industries.